S100A1 regulates neurite organization, tubulin levels, and proliferation in PC12 cells.
As a first step in determining what cellular processes are regulated by the calcium-modulated protein S100A1 isoform in neurons, the effects of ablated S100A1 expression on neurite organization and microtubule/tubulin levels in PC12 cells were examined. A mammalian expression vector containing the rat S100A1 cDNA in the antisense orientation with respect to a cytomegalovirus promoter was constructed and transfected into PC12 cells. Indirect immunofluorescence microscopy confirmed decreased S100A1 protein levels in all three stable transfectants (pAntisense clones) that expressed exogenous S100A1 antisense mRNA. In response to nerve growth factor, pAntisense clones extended significantly more neurites than control cells (4.01 +/- 0.16 versus 2.93 +/- 0.16 neurites/cell). This increase in neurite number was accompanied by an increase in total alpha-tubulin levels in untreated (4.0 +/- 0.6 versus 1.76 +/- 0.4 ng of alpha-tubulin/mg of total protein) and nerve growth factor-treated pAntisense clones (4.15 +/- 0.4 versus 2. 04 +/- 0.5 ng of alpha-tubulin/mg of total protein) when compared with control cells. At high cell densities, pAntisense clones exhibited a significant decrease in anchorage-dependent growth. In soft agar, pAntisense clones formed significantly more colonies (153 +/- 8%) than control cells (116 +/- 5%). However, the pAntisense soft agar colonies were significantly smaller than those observed in control cells (40.6 +/- 3.0 versus 59.5 +/- 1.2 micron). These data suggest that cell density inhibits both anchorage-independent and -dependent growth of pAntisense clones. In summary, ablation of S100A1 expression in PC12 cells results in increased tubulin levels, altered neurite organization, and decreased cell growth. Thus, S100A1 may directly link the cytoskeleton and calcium signal transduction pathways to cell proliferation.
Zimmer
D B
DB
Department of Pharmacology, School of Medicine, University of South Alabama, Mobile, Alabama 36688, USA. dzimmer@jaguar1.usouthal.edu
Cornwall
E H
EH
Reynolds
P D
PD
Donald
C M
CM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D002455:Cell Division / Q000502:physiology*; D003001:Cloning, Molecular; D019084:Fluorescent Antibody Technique, Indirect; D016501:Neurites; D016376:Oligonucleotides, Antisense / Q000235:genetics; D016716:PC12 Cells; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D009418:S100 Proteins; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D009418:S100 Proteins; C110294:S100A1 protein; D014404:Tubulin; D002118:Calcium